This application relates to a process for making tubulysin analogs, especially those adapted for making antibody-drug conjugates, and intermediates used in such process.
The tubulysins are cytotoxins first isolated from cultures of the myxobacteria Archangium gephyra or Angiococcus disciformis, each producing a different tubulysin mixture. They belong to a group of antimitotic polypeptides and depsipeptides that includes the phomopsins, the dolastatins, and the cryptophycins (Hamel 2002). During mitosis, a cell's microtubules reorganize to form the mitotic spindle, a process requiring the rapid assembly and disassembly of the microtubule constituent proteins α- and β-tubulin. Antimitotic agents block this process and prevent a cell from undergoing mitosis. At the molecular level the exact blockage mechanism may differ from one antimitotic agent to another. The tubulysins prevent the assembly of the tubulins into microtubules, causing the affected cells to accumulate in the G2/M phase and undergo apoptosis (Khalil et al. 2006). (Full citations for the documents cited herein by first author or inventor and year are listed at the end of this specification.)
The tubulysins have a tetrapeptidyl scaffold consisting of one proteinogenic and three non-proteinogenic amino acid subunits as shown in formula (I): N-methylpipecolinic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubuphenylalanine (Tup, R′ equals H) or tubutyrosine (Tut, R′ equals OH). The tubulysins are named A, B, and so forth, with structural variations at residues R′, R″ and R′″ of formula (I) as shown in Table I.

TABLE INaturally Occurring TubulysinsTubulysinR′R″R″′AOHOC(═O)MeCH2OC(═O)i-BuBOHOC(═O)MeCH2OC(═O)n-PrCOHOC(═O)MeCH2OC(═O)EtDHOC(═O)MeCH2OC(═O)i-BuEHOC(═O)MeCH2OC(═O)n-PrFHOC(═O)MeCH2OC(═O)EtGOHOC(═O)MeCH2OC(═O)CH═CH2HHOC(═O)MeCH2OC(═O)MeIOHOC(═O)MeCH2OC(═O)MeUHOC(═O)MeHVHOHHYOHOC(═O)MeHZOHOHHPretubulysinHHMe
The potency of the tubulysins has engendered substantial interest in using them or their analogs as anticancer agents. Consequently, there exists substantial art on the synthesis of the tubulysins or their analogs. See, for example: Cheng et al. 2013; Cong et al. 2015; Domling et al. 2010; Kazmaier et al. 2013; Park et al. 2015; Perez et al. 2015; Richter 2015; Sani et al. 2007; Shibue et al. 2010; Wipf et al. 2010; Yang et al. 2013; and Zanda et al. 2013.
A type of anticancer agent that is generating strong interest is an antibody-drug conjugate (ADC, also referred to as an immunoconjugate). In an ADC, a therapeutic agent (also referred to as the drug, payload, or warhead) is covalently linked via a linker to an antibody whose antigen is expressed by a cancer cell (tumor associated antigen). The antibody, by binding to the antigen, delivers the ADC to the cancer site. There, cleavage of the linker or degradation of the antibody leads to the release of the therapeutic agent. Conversely, while the ADC is circulating in the blood system, the therapeutic agent is held inactive because of its covalent linkage to the antibody. Thus, the therapeutic agent used in an ADC can be much more potent (i.e., cytotoxic) than ordinary chemotherapy agents because of its localized release. For a review on ADCs, see Schrama et al. 2006.
Tubulysin analogs have been proposed as the therapeutic agent in an ADC. For such use, it is necessary that a tubulysin analog have a functional group suitable for attachment of the linker. While the carboxyl group in the Tup subunit or the phenolic hydroxyl in tubulysin A can in principle serve as such functional group, a preferred functional group is a primary amine (—NH2) group, so that the linker can be attached via enzymatically cleavable peptidyl bonds. As the naturally occurring tubulysins lack a primary amine group, analogs have been made introducing such a group, especially at the 4-position of the Tup subunit. See Cheng et al. 2013; Cong et al. 2015; and Perez et al. 2015. Some of these analogs can be represented by the generic formula below, where n is 0, 1, or 2 and Ra, Rb, Rc, Rd, and Re represent either residues found in the naturally occurring tubulysins or variants thereof:
                Tubulysin Analog with 4-NH2 group in Tup subunit        
Attachment of a linker to the above analog provides a tubulysin analog/linker compound having a structure represented by formula A:

Conjugation to an antibody provides an ADC, represented by the formula below. The number of tubulysin analog molecules attached to each antibody—the drug-antibody ratio, or DAR—will depend on the conjugation chemistry, the structure of the antibody, and the structure of the linker. In the formula below, a DAR of 1 is shown for simplicity.
                Antibody-Drug Conjugate with Tubulysin Analog as Payload        
A key synthetic challenge to the preparation of such an ADC lies in the making of analog/linker A. Cheng et al. 2013 disclose a scheme entailing the coupling of a fragment B comprising the Mep, Ile, and Tuv subunits and a fragment C comprising the amine-modified Tup subunit and the linker to prepare an analog/linker A.

A specific analog/linker made by Cheng et al. 2013 is A1, whose structure is shown below. The scheme employed is recapitulated in FIGS. 1A-1B, with fragments B1 and C1 being exemplars of fragments B and C.

As can be seen from FIGS. 1A-1B, the synthesis of fragment B1 is highly linear, comprising over 12 steps, resulting in a low overall yield (estimated as 0.81% from compound 1 to compound B1 according to yields reported in Cheng et al. 2013). Further, azide 13 is potentially unstable and explosive, limiting the scheme's scalability due to safety considerations.
Thus, there is a need for a more efficient synthesis of analog/linkers A and, in particular, of fragment B, suitable for large-scale production.